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Molecular basis of plant DCL4 action that outcompetes DCL2

Nature Plants volume 12, pages 556–570 (2026)Cite this article

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A Publisher Correction to this article was published on 17 March 2026

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Abstract

Small RNAs regulate eukaryotic development and immunity. In plants, multiple DICER-LIKE (DCL) proteins produce distinct small RNAs that play diverse functions. These DCL proteins act in a hierarchical manner, with DCL4 outcompeting DCL2 being particularly important for optimal gene expression and plant growth. However, the mechanism of this hierarchical action remains unclear. Here we reveal that the second double-stranded-RNA-binding domain (dsRBD2) of DCL4 interacts with DSRNA BINDING PROTEIN 4 (DRB4), a cofactor essential for DCL4’s function. DRB4 dictates the relative biogenesis of 21- and 22-nucleotide small interfering RNAs derived from TAS loci and coding transcripts. All DCL2 proteins in seed plants lack dsRBD2; however, fusing dsRBD2 to DCL2 enhances its activity, leading to massive production of coding-transcript-derived small interfering RNAs, as well as growth defects and activated stress responses. These findings demonstrate the central role of the dsRBD2–DRB4 module, which enables DCL4 to outcompete DCL2, thereby preventing detrimental gene silencing.

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Fig. 1: The CEND is essential for the function of DCL4.
Fig. 2: dsRBD2 is required for interacting with DRB4.
Fig. 3: DRB4 dictates the competition between DCL2 and DCL4.
Fig. 4: Fusion of DCL4 CEND or dsRBD2 domains enhances DCL2 activity.
Fig. 5: Chimeric DCL2–RBD2 disrupts plant growth and induces the production of copious ct-siRNAs.
Fig. 6: Chimeric DCL2–RBD2 constitutively activates stress and immune responses.

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Data availability

The structures have been deposited in the Protein Data Bank with the accession code 9K9P. The sequencing data have been deposited in the Genome Sequence Archive (CNCB-NGDC) under accession number PRJCA032355. Source data are provided with this paper. All other data are available in the Article and Supplementary Information.

Code availability

The in-house scripts used in this study are available via Zenodo at https://doi.org/10.5281/zenodo.18411771 (ref. 91).

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Acknowledgements

We thank H. Wu, Y. Qiu, W. Luo, Y. Yan, Y. Hou and Z. Wang (SUStech) for experimental assistance and helpful discussion. This work was supported by the National Natural Science Foundation of China (grant nos 32261160572 to H.G., 32325008 to J.D. and 32400449 to W.Y.); Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture (grant no. AGIS-ZDXM202201 to H.G.); the Shenzhen Science and Technology Program (grant nos KQTD20190929173906742 to H.G. and J.D., ZDSYS20230626091659010 to H.G. and J.D., RCJC20221008092720004 to J.D., JCYJ20190809163019421 to Y.L. and JCYJ20250604144203004 to W.Y.); and the New Cornerstone Science Foundation (grant no. NCI202235 to H.G.). J.D. is an investigator at the SUSTech Institute for Biological Electron Microscopy.

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  1. New Cornerstone Science Laboratory, Shenzhen Key Laboratory of Plant Genetic Engineering and Molecular Design, Institute of Plant and Food Science, Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, China

    Yuelin Liu, Wei Yan, Qianyan Linghu, Huijuan Tan, Yajie Pan, Siqi Yan & Hongwei Guo

  2. Shenzhen Key Laboratory of Plant Genetic Engineering and Molecular Design, Institute of Plant and Food Science, Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, China

    Li Feng, Changshi Wang, Jixian Zhai & Jiamu Du

  3. Institute for Biological Electron Microscopy, Southern University of Science and Technology, Shenzhen, China

    Jiamu Du

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Contributions

H.G. conceived the project. Y.L. and H.G. designed the experiments. Y.L., Q.L., H.T. and S.Y. prepared the genetic materials. Y.L. collected the genetic phenotypes and carried out the western blot assays, northern blot assays and qRT-PCR assays. W.Y., L.F., Y.P. and J.Z. carried out the sRNA-seq and mRNA-seq bioinformatics analyses. C.W. and J.D. performed crystallization and structure determination. Y.L. and H.G. wrote the manuscript with input from the other authors. All authors contributed to manuscript preparation.

Corresponding author

Correspondence to Hongwei Guo.

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Nature Plants thanks Tuan Anh Nguyen, Sascha Laubinger and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Phylogenetic tree and conserved domain organizations of Dicer and DCL proteins across various species.

Left, a Maximum-likelihood phylogenetic tree of DCLs from representative seed plants, non-seed plants, chlorophyte, rhodophyte, and animals. Right, conserved domain organizations of the indicated DCL proteins.

Extended Data Fig. 2 DCL4 interacts with DRB4 via dsRBD2.

a, Co-IP analysis assessing the interaction between DCL4-GFP, DCL4∆CEND-GFP, and DRB4 using gD4/dcl4-2 and gD4∆CEND/dcl4-2 transgenic plants. b, Y2H assay identifying the domains of DRB4 that interact with DCL4 dsRBD2. c, GST-pull down analysis assessing the interaction between DRB4 and dsRBD1, dsRBD2, and dsRBD2mutLW, with dsRBD1 serving as a negative control. d, Protein levels of DCL4-GFP and DCL4mutLW-GFP in independent gD4/dcl4-2 and gD4mutLW/dcl4-2 transgenic lines, respectively. Black arrows indicate the expected sizes of DCL4-GFP and DCL4mutLW-GFP proteins. e, Co-IP analysis of the interaction between DRB4 and either DCL4-GFP or DCL4mutLW-GFP in transgenic plants, with DCL4∆CEND-GFP serving as a negative control. The experiments in a, c, and e were independently repeated twice, and that in d was independently repeated three times. All repeats showed consistent results.

Source data

Extended Data Fig. 3 Additional RNA gel-shift assays showing distinct dsRNA-binding affinities of DCL4, DCL4–DRB4 complex, and DCL4ΔCEND.

a, b, RNA gel-shift assays were performed with DCL4, the DCL4–DRB4 complex, and DCL4ΔCEND. Increasing concentrations of the indicated proteins (2, 4, 6, 8, 10, 12, and 16 nM) were added to dsRNA-binding reactions. The experiments were independently repeated three times with consistent results; panels a and b show the two additional independent replicates corresponding to Fig. 2c. c, The apparent dissociation constants (Kd) were calculated based on quantification of the gel-shift images in panels a and b and in Fig. 2c. Data are presented as means ± SD (n = 3 independent replicates). d, RNA gel-shift assay performed with higher concentrations of DCL4ΔCEND (10, 20, 40, 60, 80, 100, and 150 nM). e, The Kd value for DCL4ΔCEND–dsRNA interaction was calculated from quantification of the gel-shift image in panel d. Data are presented as means ± SD (n = 3 independent replicates).

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Extended Data Fig. 4 Conservation of key amino acids at the interaction interface between DRB4 and DCL4 dsRBD2 across plant species.

a, Amino acid sequence alignment of dsRBD2 from various plants. Amino acids involved in interaction with DRB4 are marked with an asterisk (*) and point mutations that disrupt this interaction are indicated by inverted triangles (▼). b, GST pull-down assay showing that DRB4 does not interact with dsRBD2 of DCL1 or DCL3, with DCL4 dsRBD2 serving as the positive control and DCL4 dsRBD1 as the negative control. c, Phylogenetic tree depicting the dsRBD1 and dsRBD2 domains from the DCL proteins across seed plants. The experiment in b was independently repeated twice, with consistent results.

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Extended Data Fig. 5 Phenotypes of gD2/dcl2-1, gD2-CEND/dcl2-1, gD2-RBD2/dcl2-1, and gD2-RBD2mutLW/dcl2-1 transgenic plants.

a, Photos of 3-week-old Col-0, dcl2-1, gD2/dcl2-1, healthy gD2-CEND/dcl2-1, healthy gD2-RBD2/dcl2-1, and gD2-RBD2mutLW/dcl2-1 transgenic plants. White triangles indicate the 5th-6th rosette leaves. The quantitative values below the photos represent the length-to-width ratio of 5th-6th rosette leaves. Data are represented as means ± SD (n = 10 rosette leaves per genotype, except gD2-CEND/dcl2-1, n = 9). Scale bar = 2 cm. b, Phenotypes of Col-0, dcl2-1, dcl4-2, gD2/dcl2-1, gD2-RBD2/dcl2-1, and gD2-RBD2mutLW/dcl2-1 plants infected with CMV. 4-week-old plants of the indicated genotypes were inoculated with CMV and grown for additional 2 weeks. Scale bar = 1 cm. White triangles indicate CMV symptoms, including small and deformed leaves that were bunched together.

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Extended Data Fig. 6 Overexpression of RBD2 alone does not markedly affect DCL4 activity.

a, Phenotypes of 3-week-old Col-0, dcl4-2, drb4-1, and 35S::RBD2-GFP/Col-0 transgenic plants. 50 transgenic plants from two independent lines were observed. White triangles indicate the 5th-6th rosette leaves. The quantitative values below the photos indicate the length-to-width ratio of the 5th-6th rosette leaves. Data are represented as means ± SD (n = 10 rosette leaves). Scale bar = 2 cm. b, Protein levels of RBD2-GFP in independent 35S::RBD2-GFP/Col-0 transgenic lines. c, Co-IP analysis assessing the interactions of GFP or RBD2-GFP with DRB4 in 35S::GFP/Col-0 and 35S::RBD2-GFP/Col-0 plants. d, Northern blot analysis of the indicated sRNA in the tested plants. U6 RNA was used as the loading control. The experiments in b and d were independently three times, and that in c was independently repeated twice. All repeats showed consistent results.

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Extended Data Fig. 7 Additional in vitro dicing assays of DCL2 and chimeric DCL2 proteins.

a, b, In vitro dicing assays were performed with DCL2, the DCL2-RBD2 + DRB4 complex, and DCL2-RBD2mutLW. The 222-nt dsRNA substrates and resulting 22-nt siRNAs diced by DCL2 and the corresponding chimeric DCL2 proteins were visualized by Cy5 fluorescence. The experiment was independently repeated three times with consistent results; panels a and b show the two additional independent replicates corresponding to Fig. 4j.

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Extended Data Fig. 8 Fusion of DCL1 and DCL3 dsRBD2 domains does not enhance DCL2 activity.

a–c, Phenotypes of 20-day-old (a), 25-day-old (b), and 35-day-old (c) Col-0, gD2/dcl2-1, gDCL2-D1RBD2-GFP/dcl2-1 (gD2-D1RBD2/dcl2-1), and gDCL2-D3RBD2-GFP/dcl2-1 (gD2-D3RBD2/dcl2-1) transgenic plants. Scale bar = 2 cm. d, Expression levels of DCL2-GFP, DCL2-D1RBD2-GFP, and DCL2-D3RBD2-GFP in transgenic lines. Black arrows indicate the expected sizes of DCL2-GFP and the corresponding chimeric DCL2 fusion proteins. e, Overall abundance of 21-, 22-, and 24-nt siRNAs derived from TAS loci in Col-0, gD2/dcl2-1, gD2-D1RBD2/dcl2-1, and gD2-D3RBD2/dcl2-1 plants. f, Abundance of transposable element (TE)-derived 21-, 22-, and 24-nt siRNAs in the indicated plants. Bars indicate the mean; dots show individual biological replicates (n = 2) for (e) and (f). g, Northern blot analysis of representative miRNA levels in the indicated genotypes. The experiment in g was independently repeated twice, with consistent results.

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Extended Data Fig. 9 Analysis of MIRNA- and TE-derived sRNAs in gD2/dcl2-1 and gD2-RBD2/dcl2-1 transgenic lines.

a, Abundance of 21-nt and 22-nt sRNAs (TPM) derived from MIRNA loci in gD2/dcl2-1 and gD2-RBD2/dcl2-1 exhibiting diverse phenotypes. b, Levels of 21-nt, 22-nt, and 24-nt siRNAs originating from TE in the indicated genotypes. Bars indicate the mean; dots show individual biological replicates (n = 2) for (a) and (b).

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Extended Data Fig. 10 Transcriptome analyses of gD2-RBD2/dcl2-1 plants exhibiting diverse phenotypes.

a, Bar chart showing the number of upregulated and downregulated genes in different phenotypes of gD2-RBD2/dcl2-1 (at least two-fold changes, FDR ≤ 0.05). b, Venn diagrams illustrating the overlap of DCL2-RBD2 dependent differentially expressed genes (DEGs) among the phenotypes. Left, upregulated genes; right, down-regulated genes. c, Heatmap displaying expression levels of DEGs in gD2/dcl2-1 and gD2-RBD2/dcl2-1 samples exhibiting distinct phenotypes (CS, PL, PS). Each sample type includes biological replicates (R1, R2). d, Principal Component Analysis (PCA) plot of four plant samples based on gene expression profiles. e, Representative GO enrichment analysis of upregulated (red) and downregulated (blue) genes for the indicated phenotypes of gD2-RBD2/dcl2-1 plants. GO enrichment analysis was performed in AgriGOv2 using a one-sided Fisher’s exact test. p-values were adjusted for multiple comparisons using the Benjamini–Hochberg procedure, and terms with FDR ≤ 0.05 were retained. The p-values and FDR-adjusted values are provided in Supplementary Table 7.

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Supplementary information

Supplementary Information (download PDF )

Supplementary Fig. 1.

Reporting Summary (download PDF )

Supplementary Table 1 (download XLSX )

Dicer and DCL protein sequences for phylogenetic tree construction.

Supplementary Table 2 (download PDF )

X-ray data collection and refinement statistics.

Supplementary Table 3 (download XLSX )

dsRBD domains of DCLs from representative seed plants.

Supplementary Table 4 (download XLSX )

Top ten sRNA libraries ranked by the percentage of 22-nt siRNAs derived from TAS loci.

Supplementary Table 5 (download XLSX )

Quantitation of 21-nt and 22-nt ct-siRNA reads in ein5, ein5 dcl4 and ein5 drb4.

Supplementary Table 6 (download XLSX )

Quantitation of 21-nt and 22-nt ct-siRNA reads in gD2 dcl2-1, gD2-RBD2 dcl2-1 (PL), gD2-RBD2 dcl2-1 (PS) and gD2-RBD2 dcl2-1 (CS) samples.

Supplementary Table 7 (download XLSX )

Gene Ontology enrichment analysis of five-week-old gD2-RBD2 dcl2-1 (PL), gD2-RBD2 dcl2-1 (PS) and gD2-RBD2 dcl2-1 (CS) compared with gD2 dcl2-1.

Supplementary Table 8 (download XLSX )

DNA oligonucleotides used in this study.

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Unprocessed western blots or gels.

Source Data Figs. 1–6 and Extended Data Figs. 3, 5, 6 and 8–10 (download XLSX )

Statistical source data.

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Liu, Y., Feng, L., Wang, C. et al. Molecular basis of plant DCL4 action that outcompetes DCL2. Nat. Plants 12, 556–570 (2026). https://doi.org/10.1038/s41477-026-02243-6

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